The Escherichia coli MFS-type transporter genes yhjE, ydiM, and yfcJ are required to produce an active bo3 quinol oxidase

Heme-copper oxygen reductases are membrane-bound oligomeric complexes that are integral to prokaryotic and eukaryotic aerobic respiratory chains. Biogenesis of these enzymes is complex and requires coordinated assembly of the subunits and their cofactors. Some of the components are involved in the acquisition and integration of different heme and copper (Cu) cofactors into these terminal oxygen reductases. As such, MFS-type transporters of the CalT family (e.g., CcoA) are required for Cu import and heme-CuB center biogenesis of the cbb3-type cytochrome c oxidases (cbb3-Cox). However, functionally homologous Cu transporters for similar heme-Cu containing bo3-type quinol oxidases (bo3-Qox) are unknown. Despite the occurrence of multiple MFS-type transporters, orthologs of CcoA are absent in bacteria like Escherichia coli that contain bo3-Qox. In this work, we identified a subset of uncharacterized MFS transporters, based on the presence of putative metal-binding residues, as likely candidates for the missing Cu transporter. Using a genetic approach, we tested whether these transporters are involved in the biogenesis of E. coli bo3-Qox. When respiratory growth is dependent on bo3-Qox, because of deletion of the bd-type Qox enzymes, three candidate genes, yhjE, ydiM, and yfcJ, were found to be critical for E. coli growth. Radioactive metal uptake assays showed that ΔydiM has a slower 64Cu uptake, whereas ΔyhjE accumulates reduced 55Fe in the cell, while no similar uptake defect is associated with ΔycfJ. Phylogenomic analyses suggest plausible roles for the YhjE, YdiM, and YfcJ transporters, and overall findings illustrate the diverse roles that the MFS-type transporters play in cellular metal homeostasis and production of active heme-Cu oxygen reductases.


Introduction
Respiratory complexes are oligomeric membrane proteins with multiple cofactors, which are widely distributed among prokaryotes and eukaryotes.Their biogenesis is an intricate process involving the insertion of appropriate cofactors into the subunits and assembly of mature subunits into functional enzymes [1][2][3].The cytochrome c oxidases (Cox) and quinol oxidases (Qox) catalyze the terminal steps of aerobic respiration, which is a four-electron reduction of oxygen to water [4][5][6].Both enzymes are multi-heme complexes containing different types of hemes (a, b, and c) [4][5][6].However, the two types of oxidases are different with respect to their electron donors as substrates.Cox employs extra-cytoplasmic water-soluble or membraneattached c-type cytochromes, whereas Qox uses lipid-soluble membrane-integral quinones.The aa 3 -type cytochrome c oxidase (aa 3 -Cox or mitochondrial complex IV) contains two atype (a and a 3 ) hemes and also two Cu centers (Cu A with two Cu atoms and Cu B with one Cu atom near of the Fe atom of heme a 3 ) [7][8][9].The cbb 3 -type cytochrome c oxidase (cbb 3 -Cox) is exclusively found in prokaryotes, and it contains three c-type (c o , c p1 and c p2 ) hemes, two btype (b and b 3 ) hemes and only one Cu atom near heme b 3 iron at the Cu B center) [10][11][12].Some Qox enzymes, like the Escherichia coli bo 3 -type Qox (bo 3 -Qox), also contain one Cu atom at their Cu B center [13] like that of the aa 3 -Cox or the cbb 3 -Cox [14,15].The nature of the cofactors, subunit structures, and electron donors vary among the heme-Cu oxygen reductases but their catalytic Fe-Cu B centers remain conserved [15,16].Besides bo 3 -Qox, E. coli has two other bd-type Qox enzymes (bd-Qox1 and bd-Qox2) involved in aerobic respiration, but they contain no Cu atom [6,17,18].
Earlier studies indicated that covalent insertion of the c-type hemes to apoproteins is carried out by the cytochrome c maturation (ccm) systems (e.g., cbb 3 -Cox) [19].The Ccm systems operate independently from the insertion of axially coordinated a-, o-, and b-type hemes [2,12].Coordination of the b-type hemes to the apoproteins may be spontaneous, like the soluble four-helical cytochrome b 562 [20].In other cases, the process might be chaperone-assisted, like the a-type hemes of aa 3 -Cox that rely on the Surf-like (Surf1 or Shy1) proteins [21][22][23][24].In Paraccocus denitrificans Surf1 [23] and in Thermus thermophilus Surf1q and CbaX [25] are essential to produce active ba 3 -type quinol oxidases (ba 3 -Qox), possibly needed for the insertion of the a-type hemes.Similarly, the insertion of the b-type hemes to the facultative photosynthetic model organism Rhodobacter capsulatus cbb 3 -Cox requires the CcoS protein [26,27].While the small protein CydX was proposed to position/stabilize the b-type hemes of the bdtype quinol oxidase [18,28,29], this process remains unknown in bo 3 -Qox.
In contrast to the heme groups, Cu insertion into the Cox enzymes has been studied in more detail.In Rhodobacter species, the mitochondrial Sco-like proteins [30] SenC or PrrC [31][32][33][34], PCuAC-like (PccA) [32,33,35], and Cox11 [36][37][38][39] chaperones are involved in this process [39][40][41][42].In the case of cbb 3 -Cox, Cu is imported by a MFS-type transporter (CcoA) and reduced via a cupric reductase (CcoG) on its way to the cytoplasm [2,43].Then, Cu is channeled through a specific chaperone (CopZ) and a P 1B -type transporter (CcoI, CtpA or CopA2) to the periplasmic Sco-like and PCuAC-like chaperones [2,26,44,45], in its way to the Cu B center of cbb 3 -Cox [2,[46][47][48].In R. capsulatus, ccoA mutants are cbb 3 -Cox Cu-deficient and unable to import radioactive 64 Cu [46,47].This cytoplasmic deficiency can be rescued either by exogenous Cu supplementation, or by deletion of the P 1B -type Cu exporter CopA, involved in excretion of excess Cu out of the cytoplasm.Remarkably, similar studies in Rhodobacter sphaeroides indicated that CcoA is solely dedicated to Cu insertion into the cbb 3 -Cox and is not required for the similar heme-Cu B center of aa 3 -Cox [49].For the eukaryotic aa 3 -Cox, Cu located in the mitochondrial intermembrane space is conveyed to the Cu A center via Cox17 [40][41][42].Although no homologue of Cox17 exists in prokaryotes, recently, the Bradyrhizobium japonicum ScoI homologue and the thioredoxin TlpA were shown to metalate in vitro the Cu A center of cognate aa 3 -Cox [50,51].Apparently, distinct Cu routes for the biogenesis of similar centers occur in species containing different types of Cox enzymes.
The superfamily of MFS-type transporters belongs to one of the largest groups of secondary active transporters and are exceptionally diverse and ubiquitous to all three kingdoms of living organisms.They selectively transport a wide range of substrates, including sugars, amino acids, peptides, and antibiotics [52].Despite their structural similarities, members of this superfamily are divided into many families and subfamilies, classified in the IUBMB-approved Transport Classification Database (TCDB, http://www.tcdb.org),based on the diversity of their substrates and their modes of transport (uniporters, symporters, and antiporters).To date, about 105 families of the MFS-type transporters are reported [53], and among them about 28 are classified as Uncharacterized Major Facilitators (UMFs).The CalT subfamily is defined based on their conserved MXXXM and HXXXM motifs [49] and phylogenetic relatedness.They also frequently co-occur with the Cox enzymes [48,49].The R. capsulatus CcoA is the founding member of this subfamily as the first bacterial Cu uptake transporter involved in the biogenesis of the cbb 3 -Cox [46], and is also the first MFS-type transporter that uses Cu as a substrate [48,49].Some CcoA-distant members (i.e., the RfnT-like proteins) of the CalT family are also Cu transporters but they do not provide Cu to the cbb 3 -Cox [48], suggesting that they might play a role in the biogenesis of other cupro-enzymes.
In this work, the role of MFS-type transporters of unknown function (UMFs) in E. coli bo 3 -Qox biogenesis was investigated employing a genetic approach.Using mutants lacking both the bd-Qox1 and bd-Qox2 enzymes, where the bo 3 -Qox was the only intact terminal oxidase, the uncharacterized MFS-type transporters YhjE, YdiM, and YfcJ were shown to be required to produce active bo 3 -Qox to support E. coli aerobic respiration.Of these UMFs, YhiE and YdiM affected cellular Fe and Cu homeostasis, respectively, suggesting that MFS-type transporters are required for the biogenesis of different heme-Cu oxygen reductases, possibly as metal or related compound transporters.

Growth conditions, strains and plasmids used
The bacterial strains and plasmids used in this work are described in S1 Table in S1 File.All E. coli K-12 strains were grown at 37˚C on Luria Bertani (LB) enriched or M9 minimal media, supplemented with ampicillin (Amp, 100 μg/ml) and kanamycin (Km, 50 μg/ml), as appropriate.For anaerobic growth, liquid cultures in filled vessels and plates placed in anaerobic jars with H 2 +CO 2 generating gas-packs (Becton, Dickinson and Co., MD) were used.The optical density (OD 600 ) of cells in liquid cultures were monitored at 600 nm.

Kan S derivatives of the MFS-type transporter mutants
The putative MFS-type transporter mutants ΔsetC, ∆yhjE, ∆yhjX, ∆ynfM, ∆ydiM, ∆yebQ, ∆yfcJ, ∆araJ and the ∆cyoB mutant were obtained from the E. coli Keio library and were Kan R [54].In each case, the kanamycin cassette was removed by introduction of the Flp recombinase carried by the plasmid pEL8 (pCP20) [55], which is Amp R and temperature sensitive (Ts) for replication.After electroporation, Amp R mutants harboring pEL8 were grown at 30˚C on LB containing ampicillin to allow excision of the kan cassette via its FRT sites located adjacent to it.Plates were transferred to 42˚C to eliminate the Amp R provided by pEL18, and the genotypes of the Kan S and Amp S colonies were confirmed by PCR using appropriate primers (S2

Construction of the Δbd-Qox1and Δbd-Qox1+Δbd-Qox2 knockout derivatives of selected MFS-type transporter mutants
The Kan S and Amp S derivatives of chosen MFS-type transporter mutants were used as recipients to knockout the bd-Qox1 and bd-Qox2 by P1 transduction.The Δbd-Qox1 derivatives of the MFS-type transporter mutants were obtained by using a P1 lysate grown on fresh cultures of the cydB:kan (Δbd-Qox1) strain, in LB medium supplemented with 0.2% glucose and 5 mM CaCl 2 .Before use, the P1 lysates were sterilized with a few drops of chloroform, and the recipient cells were mixed with the P1(cydB::kan) lysate (at 1:1 v/v ratio), incubated 20 min at 37˚C, supplemented with one volume of 1M CaCl 2 and further incubated for 40 min at 37˚C in LB medium.The Kan R (i.e., Δbd-Qox1) transductants were selected on kanamycin containing plates supplemented with 5 mM sodium citrate to chelate Ca ++ required for P1 reinfection.Following extensive purification, the genotypes of the double (i.e., ΔMFS + Δbd-Qox1) mutants were confirmed by PCR using the primers listed in S2 Table in S1 File.
To construct the triple (i.e., ΔMFS + Δbd-Qox1 + Δbd-Qox2) mutants, the Kan R marker in the cydB gene of the ΔMFS + Δbd-Qox1 double mutants was removed using pEL8 as described above.The Kan S derivatives thus obtained were used to knock out the bd-Qox2 by transduction using a P1 lysate obtained by growth on the ∆appB::kan (bd-Qox2) mutant.The triple mutants lacking both the Δbd-Qox1, Δbd-Qox2 and the desired deletion of MFS-type transporter were selected on kanamycin containing plates, and their genotypes confirmed by PCR using the primers listed in S2 Table in S1 File.

RNA isolation and RT-PCR assays
The E. coli cells used for RNA isolation and subsequent RT-PCR analyses were grown aerobically at OD 600 of 0.05, 0.1 (early growth) and 0.15 (late growth), as needed.Prior to RNA extraction, the cultures were washed with sterile water treated with two volumes of "RNAprotect Bacteria Reagent" (Qiagen).The total RNA was extracted using the Qiagen RNeasy mini kit according to the "Enzymatic Lysis of Bacteria" protocol of the manufacturer.10 μg of total RNA was digested with RNAse-free Dnase I from Qiagen for 25 min at room temperature, followed by overnight precipitation using 20 μl of NaOAc (3M, pH 5.5), 3 μl of glycogen (5mg/ ml), and 600 μl ethanol in a final volume of 800 μl. 2 ng of total RNA were used for RT-PCR analyses with OneStep RT-PCR kit from Qiagen using the CyoAQ-F/CyoAQ-R (327 bp amplicon), CyoBQ-F2/CyoBQ-R3 (322 bp amplicon), CyoCQ-F/CyoCQ-R (344 bp amplicon), and CyoDQ-F/CyoDQ-R (310 bp amplicon) primer pairs (S2 Table in S1 File) to reverse transcribe and amplify separately portions of mRNA specific of cyoA, cyoB, cyoC, and cyoD, respectively.The RrsA-F1 and RrsA-R1 primers were used as an internal control to reverse transcribe and amplify a 100 bp long portion of the 16S ribosomal mRNA.DNA contamination was checked using the master mix containing the heat-inactivated reverse transcriptase (95˚C, 15 min) prior to the RT-PCR analyses.The amplified products were separated using 2% agarose gel, and their intensities estimated using ImageJ software (NIH).

Reduced-minus-oxidized optical difference spectra
To monitor the presence of bo 3 -Qox in appropriate E. coli mutants, optical spectra of n-dodecyl β-D-maltoside (DDM)-solubilized membranes from cells grown aerobically at OD 600 of 0.1 were recorded at room temperature between the 500 and 600 nm using a Varian Cary 50 UVvisible spectrophotometer.DDM-solubilized membrane fractions (final concentration of 5 mg/mL) were prepared in 25 mM Tris-HCl pH 7.0, 150 mM NaCl and 1 mM 4-benzenesulfonyl fluoride hydrochloride (AEBSF).Reduced minus oxidized optical difference spectra were obtained by subtracting the spectra of samples fully reduced with sodium dithionite from the spectra of samples fully oxidized with potassium ferricyanide to detect the bo 3 -Qox.

Determination of the bo 3 -Qox enzyme activity
The oxygen consumption activity of bo 3 -Qox was monitored using a Clark-type oxygen electrode (INSTECH, Sys203 model).The cells were grown on LB medium to an OD 600 of 0.1, washed with 0.1 M potassium phosphate buffer, pH 7.0 and resuspended in the same buffer to a total of OD 600 of 0.5 per assay.400 μM of ubiquinol-1 was used as an artificial electron donor in the presence of 5 mM of DTT, and the electrode chamber contained one ml of the assay buffer (0.1 M potassium phosphate, pH 7.0, and 0.05% of DDM) at 30˚C.The enzymatic reaction was initiated by adding the cells.When tested for inhibitor sensitivity, cells were incubated with either 10 μM of sodium sulfide (Na 2 S) or 200 μM of potassium cyanide (KCN) for 2 min prior to addition to the reaction mixture.The μM of oxygen consumed/min/OD 600 of cells were calculated using the formula: ∆mm-Hg x 236/140/min/OD 600 of cells (140 mm-Hg corresponding to 236 μM of oxygen at 30˚C was taken as the maximum of oxygen present in the electrode chamber).
Radioactive 64 Cu and 55 Fe uptake assays using whole cells Whole cells radioactive 64 Cu uptake assays were performed according to [47].The radioactive 64 Cu (1.84 x 10 4 mCi/μmol specific activity) was obtained from the Mallinckrodt Institute of Radiology, Washington University Medical School.E. coli strains were grown at an OD 600 of 0.1 in 10 ml of LB supplemented with the appropriate antibiotics, centrifuged, washed with the assay buffer (50 mM sodium citrate, pH 6.5 and 5% glucose) and re-suspended in one ml of the same buffer.All cultures were normalized to the same number of total cells (7.5 X 10 8 cells) per 500 μl based on their OD 600 values.Cells were pre-incubated at 35˚C or 0˚C for 10 min before the assay, and the uptake activity was initiated by addition of 10 7 cpm of 64 Cu, determined immediately before use (half-life of 64 Cu isotope is ~12 h).At each time point, 50 μl of 1 mM CuCl 2 and 50 μl of 50 mM EDTA (pH 6.5) were added to an aliquot of 50 μl of assay mixture to stop the uptake reaction, and the samples were placed on ice.At the end of the assay, cells were pelleted, pellets washed twice with 100 μl of ice-cold 50 mM EDTA solution, re-suspended in 1 ml of scintillation liquid, and counted using a scintillation counter (Coulter-Beckman Inc.) with wide open window.The uptake assay with 55 Fe (1 μmol correspond to 73 mCi/mg specific activity) was performed essentially as described for 64 Cu, except that 1M sodium ascorbate was added to the 55 Fe stock solution and incubated for 10 min at room temperature to reduce it prior to the assays.The assays were stopped using 1 mM of FeSO 4 instead of CuCl 2 and processed as described for the 64 Cu uptake assays.

Statistical analyses
In all cases, at least three independent experiments were performed with at least three technical replicates.The error bars reflect the standard deviation with n indicating the number of independent repeats for each experiment.Statistical analyses were performed using the Student ttest with the wild-type activity as reference, and all p-values (when a phenotype is involved) were <0.05 as needed.

Search for distant CcoA homologues among the E. coli MFS-type transporters
Homology searches were performed to identify putative CalT family members in E. coli that contains bo 3 -Qox, but lacks cbb 3 -Cox, to inquire whether the two similar heme-Cu B center containing enzymes share analogous Cu-uptake pathways.Although CalT homologues are readily identified in species belonging to the Gammaproteobacteria [48,49], including Pseudomonas aeruginosa, Shewanella pealeana, and Vibrio species, none are found in the Enterobacteriaceae, including E. coli (EcoCyc, https://ecocyc.org).Currently there are about 70 ORFs annotated as an "MFS-type transporter" in the genomes of various E. coli strains, and about 28 of them have an unknown function (i.e., UMFs).None of these UMFs contains the conserved hallmark (membrane-integral Cu-binding motifs MXXXM and HXXXM) of the CalT family members [11].This observation suggested that cytoplasmic import of Cu inserted to the E. coli bo 3 -Qox Cu B center might be delivered by a CalT-unrelated transporter(s), like the R. sphaeroides aa 3 -Cox [49].However, this suggestion did not exclude whether any one of the UMFs could be involved in bo 3 -Qox production.Consequently, these UMFs were scrutinized by aligning their amino acid sequences with that of the canonical CalT member (i.e., R. capsulatus CcoA) and the occurrence of potential metal binding amino acid residues, like Cys, Met and His [62] (Supplementary Materials, S1 Fig) .This search yielded eight candidates, yfcJ, yhjX, yebQ, ynfM, ydiM, yhjE, araJ, and setC that were studied further.

MFS-type transporters that affect the bo 3 -Qox supported respiration in E. coli
E. coli contains three distinct terminal respiratory oxidases, the bo 3 -Qox, bd-Qox-1 and bd-Qox-2 that convert oxygen to water during respiration.The bo 3 -Qox is the major enzyme when oxygen concentration is high in the growth media, whereas bd-Qox-1 becomes predominant when the oxygen level is low [63,64].Simultaneous absence of these enzymes renders E. coli defective for respiration.However, under certain conditions such as carbon and phosphate starvation, a third O 2 reductase, the bd-Qox-2 encoded by appBCX, could be induced [65,66].The occurrence of suppressor mutations that turn on the bd-Qox-2 is frequent, and this event readily overcomes the respiratory defect of a double mutant lacking both bo 3 -Cox and bd-Qox-1 [67].Hence, assessing the role, if any, of the UMFs in the production of an active bo 3 -Cox requires an E. coli strain lacking both the bd-Qox-1 and bd-Qox-2 enzymes.Such a double mutant renders the aerobic respiratory growth of E. coli exclusively dependent on the activity of bo 3 -Qox.Thus, the double deletion ∆bd-Qox1 + ∆bd-Qox2 strain (strain BF24 with an active bo 3 -Qox) and the triple ∆bo 3 -Qox + ∆bd-Qox1 + ∆bd-Qox2 mutant (strain BF17 with an inactive bo 3 -Qox) were constructed as positive and negative controls for bo 3 -Qox activity, respectively, using the E. coli K-12 Keio collection library [54] (Materials and Methods).The deletion alleles of the desired UMFs, equally originating from the Keio library, were introduced under anaerobic growth conditions on minimal medium into the double deletion ∆bd-Qox1 + ∆bd-Qox2 background, and their aerobic respiratory growth phenotypes were determined in both minimal (M9) and enriched (LB) media.For the sake of simplicity, these mutants are referred to as bo 3 + (double mutant ∆bd-Qox1 + ∆bd-Qox2 with active bo 3 -Qox), ∆bo 3 (triple mutant ∆bo 3 -Qox + ∆bd-Qox1 + ∆bd-Qox2 with inactive bo 3 -Qox), and ∆mfs (triple mutant with a chosen ∆mfs + ∆bd-Qox1 + ∆bd-Qox2, where ∆mfs corresponds to ∆yfcJ, ∆yhjX, ∆yebQ, ∆nfM, ∆ydiM, ∆yhjE, ∆araJ, or ∆setC, as appropriate).
As expected, the bo 3 + (∆bd-Qox1 and ∆bd-Qox2) strain grew aerobically, though less vigorously than the wild-type parental E. coli K-12 (BW25113) strain (S1 strain.In contrast, the ∆yhjE (BF22), ∆ydiM (BF23) and ∆yfcJ (BF21) mutants exhibited aerobic growth defect like the ∆bo 3 strain while their anaerobic growth were fine (Fig 1, top and bottom rows).On aerobic-enriched medium, unlike the remaining ∆mfs derivatives or the bo 3 + (∆bd-Qox1 and ∆bd-Qox2) strain that can attain an OD 600 of ~4 (with 1 h doubling time), the ∆yhjE, ∆ydiM, and ∆yfcJ mutants and the ∆bo 3 strain can reach a maximum OD 600 of only ~0.15 (with ~4 h doubling time), indicating that their biomass yields were very low.The growth defect of the ∆yhjE, ∆ydiM and ∆yfcJ mutants in the absence of bd-Qox1 and bd-Qox2 suggested that these UMFs might be required to produce an active bo 3 -Qox under aerobic growth conditions.

Effects of YhjE, YdiM, and YfcJ on the transcription of bo 3 -Qox
Whether the aerobic growth defect seen in the ∆yhjE, ∆ydiM and ∆yfcJ mutants reflected the lack of transcription of the cyoABCD operon encoding the bo 3 -Qox subunits was tested.As the aerobic growth is needed to produce the bo 3 -Qox, transcription of cyoB gene by RT-PCR was used as a proxy for the cyoABCDE operon.Mutant cells grown under aerobic conditions at an OD 600 of ~0.05 and ~0.1 (early stage of growth) showed that the cyoB transcript was detectable in the ∆yhjE, ∆ydiM, and ∆yfcJ mutants (Fig 2A).However, at later growth stages (OD 600 of ~0.15 or above) where cell growth was arrested, the amounts of cyoB mRNA greatly decreased, possibly reflecting compromised mRNA transcription or stability upon growth stagnation (Fig 2B).Hence, the data indicated that at least at the early stage of growth the absence of yhjE, ydiM or yfcJ did not abolish the transcription of cyoB.Similar data were also obtained for the cyoA, cyoC and cyoD genes (S2 Fig, left lanes).Note that when these mutants were complemented with the multicopy plasmid pJRHisA overexpressing a wild type bo 3 -Qox, the cyoA, cyoB, cyoC and cyoD transcripts were detectable at all growth stages (S2

Absence of YhjE or YdiM or YfcJ affects the enzymatic activity of bo 3 -Qox
The bo 3 -Qox activities of the ∆yhjE, ∆ydiM and ∆yfcJ mutants (in the ∆bd-Qox1 ∆bd-Qox2 background) were monitored using whole cells at their early stage of growth (at OD 600 = 0.1, i. e., cyoABCDE transcript is like the parent), using a Clark-type oxygen electrode and ubiquinol-1 (UQ1) as an electron donor (Materials and Methods).Under these conditions, the bo 3 + (∆bd-Qox1 and ∆bd-Qox2) strain exhibited ~34 μmoles of O 2 consumed/min/OD 600 of cells (referred to as 100%).This activity was inhibited by the addition of 10 μM of the bo 3 -Qox specific inhibitor Na 2 S (to ~15%) or 200 μM of Cox or Qox inhibitor KCN (to ~17%) (Table 1), indicating that the measured activity was specific to bo 3 -Qox.A mutant lacking bo 3 -Qox (Δbo 3 ) had ~2% of O 2 consumption activity that decreased by one half upon addition of either Na 2 S or KCN.
In comparison, the ∆yhjE, ∆ydiM and ∆yfcJ mutants (in the bd -bo 3 + background) exhibited highly decreased activities corresponding to ~8%, 4% and 5% compared to the parental strain, and similarly, these activities were inhibited drastically by the addition of Na 2 S or KCN (Table 1).The data showed that in the absence of the MFS-type transporters YhjE, YdiM, or YfcJ the bo 3 -Qox activity was drastically reduced, consequently impairing aerobic growth in the absence of the two bd-Qox enzymes.As expected, when the ∆bo 3 strain carried a plasmid born copy of cyoABCDE (pJRhisA) (14, 68) its bo 3 -Qox activity was restored (~118.5% ± 6.12), and the bo 3 + strain carrying the same plasmid overproduced (~132.3%± 0.31) bo 3 -Qox activity compared to the bo 3 + parental wild-type strain [14,68].Increased bo 3 -Qox activity was observed in the ∆yhjE (87.0%± 5.01) or ∆ydiM (49.11% ± 2.4) or ∆yfcJ (~91.17%± 6.96) mutants when they carried the plasmid pJRHisA, and their aerobic growth defects were at least partially palliated, yielding increased enzymatic activities in all cases (Table 1).In agreement with the earlier transcription profiles, RT-PCR data also indicated that that the plasmidborne cyoABCDE sustained transcription at later stages of growth (OD 600 of 0.  were undetectable despite the presence of cyoABCD mRNA, and consistent with the absence of the enzyme activity and defective aerobic growth (Table 1).The overall data indicate that the absence of either YhjE, YdiM, or YfcJ abolishes the production of an active bo 3 -Qox enzyme when this enzyme is expressed from a chromosomal copy, whereas the effect(s) of these UMFs was still apparent but less pronounced when the cyoABCDE operon was bo 3 + strain (taken as 100%).These intensities were determined using ImageJ software (NIH).A control PCR where the reverse transcriptase enzyme was inactivated at 95˚C was performed for each total RNA extract to check for DNA contamination.Each experiment is repeated at least three times, and a representative sample is shown for each case.https://doi.org/10.1371/journal.pone.0293015.g002The bo 3 -Cox activities were measured by monitoring the oxygen consumption activities of whole cells using a Clark-type oxygen electrode.The activities were measured by incubating ubiquinol-1 (UQ1) with sodium dithionate at 30˚C prior to adding the cells (see Materials and Methods) and all the assays were performed at least three times, with the p values being <0.05 for all mutants.
a The parental bo 3 + strain exhibited ~34 mmoles of O 2 consumed/min/OD 600 of cells and taken as 100%.
b + pJRhisA refers to the complementation of various mutants with a plasmid harboring bo 3 -Qox operon [14].overexpressed from the multicopy plasmid pJRHisA.Combined with the RT-PCR assays, these results suggest that the negative impact of the ∆yhjE, ∆ydiM, and ∆yfcJ mutants on bo 3 -Qox gene expression does not fully explain the complete loss of activity of bo 3 -Qox.

Cellular 64 Cu or 55 Fe uptake by mutants lacking either YhjE or YdiM or YcfJ
The E. coli bo 3 -Qox is a heme-Cu containing enzyme, and some members of MFS-type transporters transport Cu (e.g., CalT family members) [46,47,49] or Fe [70] or siderophores [71,72].Thus, the ∆yhjE, ∆ydiM and ∆yfcJ derivatives of an otherwise wild-type strain (BW25113) were assessed for their abilities to take up radioactive 64 Cu or 55 Fe using whole cells at an early stage of their growth (OD 600 of 0.1).
In the case of Cu, E. coli wild-type cells (BW25113) showed robust, time dependent and temperature sensitive (35˚C versus 4˚C) 64 Cu uptake kinetics (Fig 4A).Under the same conditions, the ΔyfcJ and ΔyhjE mutants behaved like a wild-type strain in respect to 64 Cu uptake kinetics, indicating that the absence of YfcJ or YhjE had no effect on cellular Cu accumulation.The ΔcyoB strain exhibited reduced 64 Cu uptake kinetics, suggesting that in the absence of bo 3 -Qox, the main cupro-enzyme present in E. coli, cellular Cu accumulation decreased, possibly due to Cu homeostasis.Remarkably, the ΔydiM mutant also exhibited slow 64 Cu uptake This behavior was reminiscent to that observed with the R. capsulatus ccoA mutant that is defective in 64 Cu uptake [47].As controls, when the assays were performed at 4˚C, all strains showed greatly reduced rates of 64 Cu uptake.Fe (bottom panel) uptake kinetics (Materials and Methods) were carried out at 35˚C using whole cells grown aerobically until an OD 600 of 0.1.In each case, the uptake assays were repeated at least three times using at least three independently grown cells, and the p values were > 0.05 The ∆ydiM and ∆yhjE mutants shows lower 64 Cu (p < 0.05) and higher 55 Fe (p < 0.05) accumulations in cells, respectively. https://doi.org/10.1371/journal.pone.0293015.g004 In the case of Fe, the uptake of 55 Fe-sodium ascorbate (i.e., reduced iron) followed similar kinetics for the wild type and the DycfJ and DydiM mutants, except the ΔyhjE strain that accumulated higher amounts of cellular 55 Fe (Fig 4B).Since the assays report the net accumulation of the radioisotope used (i.e., total import minus total export during a given incubation period), the data suggested that the ΔyhjE was either overactive for import, or deficient for export, of cellular Fe leading to gradual accumulation over the time (Fig 4B).As in the case of Cu, when the Fe uptake assays were performed at 4˚C, very reduced 55 Fe uptake rates were observed.Further, when uptake assays were performed without prior incubation of 55 Fe with sodium ascorbate (i.e., with oxidized form of 55 Fe), then all strains including the ΔyhjE exhibited comparable 55 Fe uptake activities.Thus, YhjE affected the transport of reduced, but not oxidized, form of Fe.Overall, whole cells uptake kinetics indicated that the absence of YdiM and YhjE perturbs Cu and Fe homeostasis, respectively, in E. coli cells.How the cellular imbalance of Cu or Fe in mutants lacking these two MFS-type transporters is linked to the observed bo 3 -Qox deficiency and aerobic growth defect, requires further studies.

YhjE is related to the putative hydroxy-ethyl-thiazol (HET) and other transporters that cluster with bo 3 -Qox
YhjE (TC: 2.A.1.6.10)belongs to a large subfamily of the MFS-type transporters with homologues in most major bacterial phyla.In TCDB, YhjE is listed as a metabolite:H+ symporter (MHS) family member, but its metabolite cargo is unknown.YhjE homologues are identified in beta-and alpha-proteobacteria in addition to gamma-proteobacteria, with sequence similarity hits (based on top 1000 blastP hits against UniProt reference proteomes) being predominately related to proteins from the Proteobacteria (49%) and Actinobacteria (43%).A sequence similarity network analysis indicates that of the previously published MFS-type transporters, YhjE is most similar to ThiU [73] (TC 2.A.1.6.12;putative thiazol transporter according to TCDB) Based on previous gene clustering and phylogenetic profiling analyses, ThiU is predicted to be a hydroxy-ethyl-thiazole (HET) transporter required for thiamin biosynthesis [73], although this hypothesis has not been tested experimentally.Based on phylogenetic reconstruction, ThiU and YhjE may be paralogs, suggesting that ThiU and YhjE could have separate functions (Fig 5A).As an example, the Haemophilus influenzae genome encodes a ThiU ortholog (HI_0418) located in the thiamine-related genes cluster, and a YhjE ortholog (HI_0281) next to two genes encoding enzymes involved in menaquinone biosynthesis (2-succinyl-6-hydroxy-2, 4-cyclohexadiene-1-carboxylate synthase/ 2-oxoglutarate decarboxylase (MenD; HI_0283) and menaquinone-specific isochorismate synthase (MenF; HI_0285).Intriguingly, MenD is a thiamine-dependent protein.(Fig 5A).However, this proximity between a gene encoding YhjE-like proteins and a gene encoding MenD is only observed in Haemophilus species.
Noticeably, YhjE-like proteins are identified in some Proteobacterial genomes that are encoded by genes next to the cyoABCD operons, encoding the structural subunits of bo 3 -Qox (Fig 5B).Except for the gene clusters from Thiotrichales, Chromatiales, and Hyphomicrobiales, where the yhjE-like gene is in the same operon with cyoABCD, most yhjE-like genes are found in the opposite orientation, suggesting that although yhjE and cyoABCD may not form an operon, still they might be co-regulated (Fig 5C).These YhjE-like proteins, although not connected to the main sequence similarity network cluster (i.e., have an E value > 1E-70 with any other protein in the main cluster), might be closely related to YhjE in the phylogenetic tree (Fig 5B).This observation suggests that the role of YhjE in bo 3 -Qox function is likely conserved outside of E. coli.

The ydiM-like genes are linked to the shikimate pathway
In the E. coli K-12 genome, ydiM (TC: 2.A.1.15.12) is located next to its paralog ydiN (TC: 2. A.1.15.13), and two other genes encoding two enzymes in the shikimate pathway, aroD and ydiB, encoding 3-dehydroquinate dehydratase and quinate/shikimate dehydrogenase enzymes, respectively.The shikimate pathway is a major link between carbohydrate metabolism and the biosynthesis of aromatic compounds via chorismate, a precursor of aromatic amino acids phenylalanine, tyrosine, and tryptophan.The cargo of YdiN is not listed in TCDB, but it was previously hypothesized to transport a shikimate by-product [74] based on the genomic context and co-expression data of the ydiN, aroD, and ydiB genes.YdiM is listed as a putative isoprenol exporter due to increase susceptibility that it provides to E. coli upon its deletion [75].
Phylogenetic and genomic context analyses of YdiM and YdiN homologues further defined their relationship to the shikimate pathway.The YdiM and the YdiN orthologous group are largely limited to Enterobacterales genomes and not widespread in Proteobacteria.YdiM/ YdiN-like proteins are frequently found in Firmicute genomes, represented by YfkL in Bacillus subtilis (Fig 6).Accordingly, among YdiM homologues (top 1000 blastP hits) ~57% are from Firmicutes and ~23% are from Proteobacteria.Remarkably, ~75% of YdiM/YdiN homologues analyzed here are encoded by a gene that is adjacent (on either the 5' or 3' side) to a gene encoding an enzyme in the shikimate pathway (Fig 6).Moreover, this frequency increases to ~86% when a larger (20 instead of the usual 10) genes window is used, showing a clear link between the YdiM and YdiN subfamily of the MFS-type transporters and the enzymes of the shikimate pathway (Fig 7).In addition to E. coli, other Enterobacteriaceae genomes including Shigella flexneri, Salmonella typhimurium, and Citrobacter tructae also

Bioinformatic analysis of YfcJ-like proteins
Currently little is known about YfcJ (TC: 2.A. 1.46.6) and its homologues.The closest related protein with some associated experimental data is YhhS (TC: 2.A.1.46.7), a paralog of YfcJ in E. coli, which was previously linked to cellular arabinose levels [77] and glyophosate (inhibitor of 5-enolpyruvylshikimate-3-phosphate synthase) resistance [78] based on loss-of-function and gain-of-function experiments, respectively.The sequence similarity network and phylogenetic reconstruction analyses were able to distinguish the YfcJ-like homologues from the closest subfamily composed of YhhS homologues.(Fig 8A and 8B).The YfcJ-like subfamily was mainly identified in Proteobacteria and Bacteroidetes (89% and 8.5% of the homologs, respectively), and within the Proteobacteria, there was a roughly equal split between gamma-(30%), alpha-(30%), and beta-(27%) proteobacteria.Analysis of conserved gene proximity revealed multiple putative operons encoding YfcJ-and YhhS-like transporters.Although defined biochemical functions could readily predicted for proteins encoded by genes neighboring the yfcJ homologues, such as amidohydrolases or tautomerases, no specific pathway or process that may be associated with YfcJ could be predicted (Fig 9, upper part).Note that the clusters 4, 5, and 12 of YhhS homologues are in putative operons with nucleotide metabolism and tRNA-related proteins, linking the YhhS family to nucleotide metabolism and tRNA modification processes based on conserved gene proximity (Fig 9, lower part).Of these clusters, the genes in cluster 4, which is dominated by Actinobacteria, are often in a putative operon with a YacP-like endoribonuclease, and a protein resembling an epoxyqueuosine reductase responsible for a synthesis of queuosine found in some tRNAs.Cluster 5 from Proteobacteria is found in a putative operon with proteins involved in nucleotide metabolism, including a putative hydrolase from the YjjG superfamily involved in cleaving nucleotides with noncanonical nucleotide bases.In cluster 12 the YfcJ-and YhhS-like homologues are in a putative operon with glutamyl-Q tRNA (Asp) synthetase, which is a protein that functions immediately downstream of epoxyqueuosine reductase involved in the synthesis of the hypermodified base glutamyl-queuosine [79].For cluster 2 in the network, which is largely confined to Actinobacteria, all YfcJ-and YhhS-like homologues are encoded by a gene that is potentially in a biosynthetic gene cluster for an unknown secondary metabolite, suggesting that they may be metabolite transporters.

Discussion
The MFS-type transporter CcoA (TC: 2.A.1.81.1) of the CalT subfamily is a well-established Cu importer identified in bacteria and required for biogenesis of the cbb 3 -Cox Cu B center [11,46,49].However, although CcoA is widespread among alpha-proteobacterial species and it frequently co-occurs with the genes encoding aa 3 -Cox and cbb 3 -Cox [48,49], it is only required for cbb 3 -Cox and not the quasi-identical Cu B center containing aa 3 -Cox, as seen with R. sphaeroides [49].Moreover, no functional ortholog of R. capsulatus CcoA is found among the ~70 MFS-type transporter genes of E. coli, suggesting a different mechanism for Cu import and biogenesis for the bo 3 -Cox Cu B center.These observations point out the specificity of the CalT members among the MFS-type transporters and indicate the possible occurrence of different routes for the biogenesis of Cu B centers of heme-Cu enzymes (e.g., E. coli bo 3 -Cox).Indeed, bacterial cbb 3 -Cox and aa 3 -Cox require specific transporters and chaperones for the biogenesis of their Cu B centers assembly, including the periplasmic Sco-like [31,35] and PCuAC-like chaperones [2,32,33,38].Moreover, cbb 3 -Cox requires in addition to the Cu importer CcoA [46] the cupric reductase CcoG [43], and the P 1B -type transporter CcoI/CtpA [26,44,45].Remarkably, none of the latter proteins are involved in the case of the aa 3 -Cox, which instead uses the Cu chaperone Cox11 [36][37][38].How the Cu B center insertion occurs in E. coli bo 3 -Qox is not known, and as a true CalT homologue does not seem to exist in this species, raising the issue of whether any other type of MFS-transporter might accomplish this function.
A survey of the E. coli genome indicated that among the ~70 MFS-type transporters, ~28 of them (i.e., UMFs) had no identified cargo, and at least eight of them were richly endowed with plausible metal-coordinating amino acid residues.This enticed us to examine the role of these UMFs in bo 3 -Cox biogenesis, using a genetic screen based on the essentiality for aerobic respiratory growth sustained by this enzyme in the absence the bd-Qox1 and bd-Qox2.This screen identified YhjE, YdiM, and YfcJ as required MFS-type transporters for bo 3 -Cox dependent aerobic respiratory growth of E. coli.In the absence of any one of these proteins, the bo 3 -Qox activity and its b-and o-type hemes were absent, even though at low cell-densities detectable amounts of cyoABCD mRNA transcripts were produced.Remarkably, a multicopy plasmid carrying these genes and overproducing the bo 3 -Qox could bypass at least partially the need for these UMFs.These findings suggested that some regulatory event(s) (e.g., titrating out a regulator) controlling the transcription or destabilizing the transcript(s) might occur in the absence of these UMFs.Alternatively, although these mutants might produce the structural constituents of the bo 3 -Qox, they could not assemble an active enzyme in the absence of the imported/exported cargo(s).Thus, the specific nature(s) of currently unidentified cargos transported by these MFS-type transporters seem important for bo 3 -Qox biogenesis.Earlier genetic studies have suggested that yhjE, ydiM, and ycfJ may be involved in transporting an unknown metabolite (see TCDB), isoprenol [75] and arabinose [77], respectively.Here, the whole-cell transport assays further showed that cells without YdiM accumulated less 64 Cu, and those without YhjE contained more reduced 55 Fe (Fig 4), while no such difference was seen in the absence of YfcJ.Note that currently no conclusive data exist for any of these transporters, as none of them has been purified and shown to bind and transport their putative substrates.
Bioinformatics analysis have been performed to unravel the function of YfcJ, YhjE and YdiM.No link between bo 3-Qox and metal transport were found for YfcJ.YhjE was referred to as a member of the metabolite: H+ symporter (MHS) Family (see TCDB), and phylogenomic analyses show that in many bacterial genomes, yhjE gene clusters with the bo 3 -Qox structural genes cyoABCD, suggesting that the role of YhjE-like transporters in bo 3 -Qox function could be widely conserved (Fig 7).Based on an analysis of previously published high-throughput (HTP) interaction data [80], YhjE was found to physically interact with FhuA, a ferrichrome outer membrane transporter [81].Out of 331 identified genetic interactions in a separate study, a positive genetic interaction was identified with fhuA (i.e., the double yhjE fhuA mutant grew better in rich medium than the single mutants), and negative genetic interactions with other Fe transporters (fecA, fecB, fecC, fecD, fepA, fepB, febD, fes, and fhuC) [80].If these results obtained with HTP studies are not misleading false positives, they could potentially explain the Fe-homeostasis defect in the ∆yhjE strain.A negative genetic interaction was also observed between yhjE and cyoA or cyoB and a positive genetic interaction with cyoC.Such results may suggest that YhjE could have functional roles beyond bo 3 -Qox biogenesis.Overall, the available experimental and bioinformatic data support that this transporter is required to produce an active bo 3 -Qox, but the underlying molecular link(s) remains unknown.
YdiM was initially selected as a candidate metal transporter based on the presence of M 21 XXXXM 26 and M 76 XXM 79 XXXM 83 motifs in its predicted TM1 and TM3, and other motifs in the TM4, and TM6 (S5 Fig) .The 3D structural model of YdiM is reminiscent of that of CcoA since the Met residues are positioned in a similar fashion throughout the TMs of both proteins.However, the putative transmembrane metal-binding motif(s) are different from the CalT subfamily members (S5 Fig) [11].These putative metal binding residues combined with the experimental data presented here suggest that YdiM could be a plausible candidate for Cu transport.In the E. coli K-12 genome ydiM gene and its paralog ydiN are clustered together with several genes involved in the shikimate pathway, which is the metabolic pathway governing biosynthesis of aromatic amino acids, like phenylalanine, tyrosine, and tryptophan [82,83].Phylogenetic analyses of bacterial species other than E. coli also indicate that ydiM and ydiN cluster frequently with the shikimate pathway genes (Figs 5 and 6).An earlier study indicated that the 3-deoxy-D-arabino-hepulosonate-7-phosphate synthase (DAHP synthase) catalyzing the first step of this pathway binds Cu, suggesting that the DAHP synthase may be a cuproenzyme [84].However, no conclusive study has been conducted, leaving the identity of the metal of DAHPS contested [85], and the link between Cu, the shikimate biosynthetic pathway, and bo 3 -Qox remains unclear, deserving future studies.
In summary, this study unexpectedly implicated three MFS-type transporters, YhjE, YdiM and YfcJ in the production of an active bo 3 -Qox in E. coli.Available data showing impaired Cu and Fe uptake kinetics suggest that YdiM and YhjE are involved in cellular metal homeostasis, which may be essential for the biogenesis of the heme-Cu enzyme bo 3 -Cox.However, the cargo of these transporters being currently unknown, and their role(s) in specific metabolic pathway(s) undefined, a direct mechanistic link between them and the expression or assembly of the bo 3 -type Qox remains hypothetical until such data become available.Nonetheless, the overall findings increased the arsenal of the different gene products that cells use to produce heme-Cu enzymes, including the bo 3 -Qox.These studies also illustrated how broad a biological function the MFS-type transporters may play in cells and spur future investigations to identify the transported substrates and shed light to the mechanistic link(s) between these MFStype transporters and the biogenesis of heme-Cu containing metalloproteins.

Fig 2 .
Fig 2. Effects of the absence of YfcJ, YhjE, and YdiM on the transcription of bo 3 -Qox.One step RT-PCR was performed on total RNA extract from bo 3 + strain as well as ∆bo 3 (cyoB), ∆yfcJ, ∆yhjE, and ∆ydiM mutants using the cyoB primers to amplify a 322 bp DNA fragment, and the rrsA primers to amplify a 100 bp region of the 16S ribosomal mRNA as a control (Materials and Methods).(A) Early stage of growth.The cells were grown under aerobic conditions at OD 600 of 0.05 (top panel) and 0.1 (bottom panel) where cell division continues.The transcription of cyoB was readily detected and showed no difference at both OD 600 .(B) Late stage of growth.The cells were grown under aerobic conditions at OD 600 of 0.15 (maximum OD 600 reached).The transcripts of cyoB gene were barely detectable when the bo 3 -Qox or the MFS-type transporters YfcJ, YhjE, and YdiM are absent.The numbers below each panel indicate the intensities of the corresponding bands, normalized to that of rrsA then compared to that seen with the

Fig 3 .
Fig 3. b-type heme compositions of the ∆yfcJ, ∆yhjE, and ∆ydiM mutants.The reduced minus oxidized spectra of the membranes prepared from the bo 3 + strain as well as the ∆bo 3 , ∆yfcJ, ∆yhjE, and ∆ydiM mutants grown under aerobic condition at an OD 600 of 0.1.The bd-Qox1 and bd-Qox2 being absent, the observed broad peak at 560 nm in bo 3 + and the strain overproducing bo 3 -Qox (bo 3 + + pJRHisA) was taken as corresponding to the hemes b and o 3 of bo 3 -Qox.This peak is drastically reduced in ∆bo 3 as well as the ∆yfcJ, ∆yhjE, and ∆ydiM strains.Each experiment is repeated at least three times, and a representative sample is shown for each case.https://doi.org/10.1371/journal.pone.0293015.g003

Fig 5 .
Fig 5. Sequence similarity analysis of YhjE-like transporters and gene neighborhoods containing its homologues.(A) Sequence similarity network using an alignment score of 110.Sequences for the network were collected by searching against UniRef50 (500 hits) with YhjE and mapping to UniRef90 for network construction.Experimentally characterized proteins or proteins with predicted functions (in italics) based on bioinformatic analyses are labeled.The taxonomic classification of each node is colored according to the key shown on top left.(B) iqTREE using edited MAFFT alignment based on UniRef50 sequences, and (C) examples of gene neighborhoods containing a YhjE-like MFS transporter with the key defining them located at the bottom right.https://doi.org/10.1371/journal.pone.0293015.g005

Fig 6 .
Fig 6.Phylogenomic analysis of the YdiM/YdiN subfamily.(A) Phylogenetic tree of YdiM, YdiN, and their YdmiM-like and YdiN-like homologues.The taxonomic classification of each leaf, presence of the chorismite mutase fusion, and whether the corresponding gene is next to a gene encoding a shikimate pathway enzyme are indicated with inner rings according to the key shown at the bottom of the figure.Lines colored by taxonomic classification connecting two leaves are used to indicate that those two proteins are encoded by the same genome.The innermost grey ring corresponds to the clusters depicted in panel B, and YfkL indicates the homologue present in Bacillus subtilus (Bs).Gene neighborhoods from clades with background shading are shown in Fig 6.Sequences are the 250 best hit from blastp against UniProt reference proteomes.The 10 most similar proteins to YdiM in E. coli, Clostridioides difficile, Klebsiella pneumoniae, Bacillus subtilis were used as an outgroup to root the tree.(B) Sequence similarity network (SSN) of YdiM/YdiN homologues.Nodes are colored by taxonomy according to the key shown at the bottom of the figure, and clusters are labeled as in the innermost grey ring of panel A. Edge-weighted Spring Embedded Layouts using % id for clustering.(C) SSN as in panel B but colored based on presence of neighbor gene(s) encoding enzyme(s) in the shikimate pathway, and (D) shows the nodes in red representing the YdiM orthologs with a chorismate mutase fusion.https://doi.org/10.1371/journal.pone.0293015.g006

Fig 7 .
Fig 7. Gene neighborhoods containing ydiM/ydiN homologues.(A) Gene neighborhoods of the YdiM and (B) gene neighborhoods of the YdiN clades.(C) Representatives from Cluster 4 shown in Fig 5. (D) Alphafold prediction depicting the YdiM-chorismate mutase fusion from Fructobacillus durionis, showing its two distinct domains.(E) Shikimate pathway from quinate to 4-hydroxy-phenylpyruvate and the structural genes of the enzymes involved.A star indicates the step catalyzed by chorismite mutase (CM) that is sometimes found fused to YdiM as shown in (D).The number of times a YdiM/YdiN homologue is encoded in a gene neighborhood with a gene encoding the indicated enzyme is shown as a heatmap (176 red to 35 blue).https://doi.org/10.1371/journal.pone.0293015.g007

Fig 8 .
Fig 8. Sequence similarity analysis of YfcJ homologues.(A) Sequence similarity network using an alignment score of 80. Sequences for the network were collected by searching against UniProt using YfcJ as a query.Clusters with examples of gene proximity in Fig 9 are circled and labeled.The taxonomic classification of each node is colored according to the key shown at the bottom left of the figure.(B) Protein sequences from the network were mapped to UniRef50 and representative nodes were used to build a phylogenetic tree.The background color of each leaf is colored according to the key.In addition to clear separation from the YhhS-like clade, the YfcJ group can be distinguished into two major clades, indicated as YfcL-like clade 1 and clade 2. https://doi.org/10.1371/journal.pone.0293015.g008

Fig 9 .
Fig 9. Conserved gene proximity analysis of YfcJ homologues.Examples of conserved gene neighborhoods encoding proteins from the YfcJ sequence similarity network are shown.Clusters corresponding to genes involved in nucleotide metabolism and tRNA modification (YhhSlike).tRNA modification (cluster 4), nucleotide metabolism (cluster 5) and tRNA modification (cluster 12) are shown with the related enzymes found shown on the right.https://doi.org/10.1371/journal.pone.0293015.g009